Microscopy of single F_oF₁‐ATP synthases— The unraveling of motors, gears, and controls

Abstract

Optical microscopy of single F 1 -ATPase and F o F 1 -ATP synthases started 15 years ago. Direct demonstration of ATPdriven subunit rotation by videomicroscopy became the new exciting tool to analyze the conformational changes of this enzyme during catalysis. Stimulated by these experiments, technical improvements for higher time resolution, better angular resolution, and reduced viscous drag were developed rapidly. Optics and single-molecule enzymology were entangled to benefit both biochemists and microscopists. Today, several single-molecule microscopy methods are established including controls for the precise nanomanipulation of individual enzymes in vitro. F€ orster resonance energy transfer, which has been used for simultaneous monitoring of conformational changes of different parts of this rotary motor, is one of them and may become the tool for the analysis of single F o F 1 -ATP synthases in membranes of living cells. Here, breakthrough experiments are critically reviewed and challenges are discussed for the future microscopy of single ATP synthesizing enzymes at work. V C 2013 IUBMB Life, 65(3): [227][228][229][230][231][232][233][234][235][236][237] 2013

Structural Asymmetry and Kinetic Limping of Single Rotary F-ATP Synthases

“…On the other hand, in human mitochondrial F-ATP synthases, and perhaps in all F-ATP synthases of eukaryotic mitochondrial origin as well, a third sub-step was found and associated with the release of Pi [82]. The details of the rotation mechanism have been reviewed elsewhere [2,3,5,6,7,8,83,84]…”

Analyzing conformational dynamics of single P-glycoprotein transporters by Förster resonance energy transfer using hidden Markov models

“…Manual assignment of FRET levels unraveled faster NBD dynamics during drug transport than in the absence of ATP or inhibited by vanadate [18, 19]. In contrast to manual analyses of FRET levels and dwell times to study the catalytic processes in F o F 1 -ATP synthase[24, 25, 27, 56], an obvious number of FRET levels and a sequence of FRET levels associated with drug transport could not be assigned for Pgp by visual inspection.…”

Imaging cytochrome C oxidase and FoF1-ATP synthase in mitochondrial cristae of living human cells by FLIM and superresolution microscopy

“…Three synchronized TCSPC cards (SPC 153, Becker&Hickl) recorded the photons [24][25][26] . FLIM and time-resolved anisotropy images were analyzed using the SPCImage software (Becker&Hickl), whereas intensity-based anisotropy imaging was analyzed using counter cards (National Instruments) and custom Matlab scripts (Mathworks) [27][28][29][30][31][32][33][34][35] .…”